Anion exchangers (AE) are a widely distributed family of proteins which regulate intracellular pCl, pH and volume in mammalian cells. Tissue-specific AE isoforms participate in the control of acid-base homeostasis in diverse organs such as bone, blood, kidney, stomach, brain and heart, and may be related to pathogenesis of several diseases including osteoporosis, stroke, ulcer and renal failure. In at least some tissues, linkage of anion exchangers to the membrane cytoskeleton contributes in the morphogenesis of the plasma membrane. We have cloned a family of tissue-specific anion exchangers, and demonstrate that they share the ability to catalyze Cl--HCO3- exchange. The studies proposed herein are focused primarily, but not exclusively, on the structure and function of the neuronal anion exchanger, AE3. These studies will address the following six specific hypotheses: (1) the neuronal anion exchanger, AE3, plays a critical role in maintaining Cl- and H+ balance in specific classes of CNS neurons; (2) this activity is under tight metabolic regulation; (3) AE3 interacts with the neuronal plasma membrane cytoskeleton and this interaction contributes to the organization of neuronal structure; (4) the N-terminal domains mediate interaction with cytoskeleton, but play a role in anion exchanger regulation and biosynthesis; (5) interaction between anion exchangers and cytoskeleton occurs at an early stage of protein biogenesis; and, (6) alternately spliced isoforms of AE3 are expressed as proteins in a cell-type specific fashion and assemble into high molecular weight complexes of unknown function. To test these hypotheses we will: (1) map the cellular and subcellular site of AE3 spliceoforms by light and electron microscopy; (2) investigate the molecular basis of AE3 regulation and its role in maintaining neuronal ionic homeostasis by site-directed mutagenesis and in situ measurement of AE3 activity, respectively; (3) identify the ankyrin binding site on anion exchangers using site-directed mutagenesis and the intracellular compartment in which the interaction occurs by pulse-chase labeling and complex analysis; and, (4) identify the cytoskeletal components, including ankyrin that associate in with AE3 spliceoforms in neurons by direct binding assay and analysis of high molecular weight protein complexes.